Generation of human cerebral organoids with a structured outer subventricular zone

Outer radial glia (oRG) emerged during mammalian evolution as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortex development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX and closely matching human oRG in vivo. In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF-producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids. Overall design: Cerebral organoids were treated with LIF or used to generate assembloids by fusion with pericyte spheroids. Single cell suspensions were obtained through papain dissociation at days 40, 60, 65 or 100 and used for single cell RNA-sequencing.

外部放射状胶质细胞（outer radial glia, oRG）在哺乳动物演化过程中作为皮层祖细胞出现，可直接支撑扩张的外侧脑室下区（outer subventricular zone, oSVZ）的发育，进而推动新皮层的扩张。体外获取外部放射状胶质细胞，对于建模并探究人类新皮层发育与扩张的潜在机制至关重要。

本研究通过白血病抑制因子（Leukemia Inhibitory Factor, LIF）激活信号转导与转录激活因子3（Signal Transducer and Activator of Transcription 3, STAT3）通路——该因子在引导型皮层类器官中无法自然产生——开发了一套源自人类多能干细胞（human pluripotent stem cells, hPSCs）的大脑类器官分化方法，该方法可重现祖细胞库向外侧脑室下区的扩张过程。

该结构化的外侧脑室下区由表达特定外部放射状胶质细胞标志物的祖细胞构成，涵盖胶质纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）、白血病抑制因子受体（Leukemia Inhibitory Factor Receptor, LIFR）及同源盒蛋白HOPX（Homeodomain-only protein HOPX, HOPX），且在体内与人类外部放射状胶质细胞高度相似。在此微环境中，皮层神经元展现出更快的成熟速率，同时代谢与功能活性显著增强。

将人类多能干细胞来源的、可分泌白血病抑制因子的脑血管周细胞（pericytes）整合至大脑类器官中，可模拟白血病抑制因子处理的效果。本研究数据表明，包含脑血管细胞类型在内的皮层微环境的细胞复杂性，有利于外部放射状胶质细胞的产生，并为在人类多能干细胞来源的大脑类器官中常规研究外部放射状胶质细胞提供了标准化实验平台。

整体实验设计：大脑类器官或经白血病抑制因子处理，或通过与周细胞球融合构建组装类器官（assembloids）；在培养第40、60、65或100天，通过木瓜蛋白酶解离获取单细胞悬液，用于单细胞RNA测序。